Method for Making Acylated Polypeptides

ABSTRACT

The present invention related to a method of producing polypeptides in transformed host cells by expressing a precursor molecule of the desired polypeptide which are to be acylated in a subsequent in vitro step. The invention is also related to DNA-sequences, vectors and transformed host cells for use in the claimed method. Further, the present invention is related to certain precursors of the desired polypeptides and certain acylation methods. The invention provides a method for making polypeptides being preferentially acylated in certain lysine ε-amino groups.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/205,110, filed Jul. 24, 2002, which claims priority under 35 U.S.C.119 of Danish application no. PA 2001 01141 filed on Jul. 24, 2001, andU.S. application No. 60/310,793 filed on Aug. 8, 2001, the contents ofwhich are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is related to a method of producing polypeptidesin transformed host cells by expressing a precursor molecule of thedesired polypeptide which is to be acylated and subsequently cleaved ata Lys cleavage site in a subsequent in vitro step. The invention is alsorelated to DNA-sequences, vectors and transformed host cells for use inthe claimed method. Further, the present invention is related to certainprecursors of the desired polypeptides and certain acylation methods.

BACKGROUND OF THE INVENTION

Recombinant DNA technology has enabled expression of foreign(heterologous) polypeptides in microbial and other host cells. In yeastexpression of heterologous polypeptides after transformation of yeastcells with suitable expression vectors comprising DNA sequences codingfor said polypeptides has been successful for many species ofpolypeptides, such as insulin and insulin precursors, glucagon, glucagonlike peptides and analogues thereof.

A common problem with expression of proteins or polypeptides of alimited size in a recombinant host is, however, proteolytic degradationof the expressed product by proteolytic enzymes produced by the hostorganism.

Thus, the isolated product may be a heterogeneous mixture of species ofthe desired polypeptide having different amino acid chain lengths.Another problem encountered in production of heterologous polypeptidesin yeast may be low yield, presumably due to proteolytic processing bothin intracellular compartments and at the plasma membrane caused byaberrant processing at internal sites in the polypeptide. Yeast containsa number of proteases used for processing yeast proteins e.g. Kex2p andYps1p which cleave at the C-terminal side of a dibasic amino acidsequence, and the carboxypeptidase Kex1p which digests remaining basicamino acids after the endoproteolytic digestion by Kex2p, and Ste13p orDap2p which cleave at X-Ala or X-Pro.

Some polypeptides, e.g. polypeptides having from about 10 to about 100amino acids chains and none or a few disulphide bonds and/or are rich inbasic amino acids, such as β-endorphine, glucagon and glucagon likepeptides may be especially susceptible to intracellular andextracellular proteolytic degradation when expressed in a transformedhost cell due to their short-chain open and non-disulfide stabilizedstructure resulting in an inhomogeneous product which may beproteolytically degraded in the N- and C-terminal ends as well asendoproteolytically degraded.

Furthermore, N-terminal cleavage of expressed polypeptides by host cellproduced enzymes may cause decreased yield of a desired product withcorrect N-terminal if the N-terminal of the expressed productconstitutes a cleavage site for endogenous enzymes. In yeast for examplethe enzyme Ste13p cleaves at X-Ala or X-Pro, where X can be any aminoacid residue. Thus, polypeptides with an Ala or Pro residue as thesecond residue from the N-terminal end may be cleaved at the N-terminalend and the recovered polypeptide may be a mixture of differentdegradation products complicating the recovery process and reducing theoverall yield.

Furthermore, small polypeptides with no or little tertiary structure andlow content of α-helixes may have a higher tendency to form β-sheetsthat stack on each other and form fibrils during fermentation and downstream separation and purification steps in large scale production.Formation of fibrils may cause unwanted precipitation with loss of thedesired product. Fibrillation may be prevented by treatment at high pH.However, such alkaline treatment is pretty harsh to the product and maycause unwanted formation of D-amino acids residues.

Human GLP-1 is a 37 amino acid residue peptide originating frompreproglucagon which is synthesised in the L-cells in the distal ileum,in the pancreas and in the brain. Processing of preproglucagon to giveGLP-1₍₇₋₃₆₎amide, GLP-1₍₇₋₃₇₎ and GLP-2 occurs mainly in the L-cells.Both GLP-1 and GLP-2 has an Ala as the second amino acid residue fromthe N-terminal end and are thus prone for N-terminal cleavage whenexpressed in a host organism such as yeast.

Introduction of lipophilic acyl groups in naturally occurring peptidesor analogues thereof has shown to lead to acylated peptides which have aprotracted profile relative to the native peptide or unmodifiedanalogues. This phenomenon is disclosed and demonstrated in WO 98/08871which discloses acylation of GLP-1 and analogues thereof and in WO98/08872 which discloses acylation of GLP-2 and analogues thereof.

When acylating precursor molecules comprising a Lys cleavage siteacylation of the Lys cleavage site should be avoided as such acylationwill prevent the subsequent cleavage. The method according to thepresent invention is a solution to this problem as it will appear fromthe following.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention is related to a method ofproducing polypeptides in transformed host cells by expressing aprecursor molecule of the desired polypeptide said precursor moleculecomprising an N-terminal extension which allows for preferentialacylation of the expressed precursor molecule and protects the expressedprecursor molecule against proteolytic degradation within the host cellor in the culture medium. In addition, the precursor molecule is easierto purify and has a less tendency to form fibrils thus allowing moreflexibility when selecting down stream separation and purification stepsin large scale operations.

In one aspect the present invention is related to a method for making apolypeptide comprising at least one lysine residue being acylated in itsε-amino group, said method comprising the following steps:

-   -   (i) culturing a host cell comprising a polynucleotide sequence        encoding a precursor molecule of the desired polypeptide under        suitable conditions for expression of said precursor molecule,        the precursor molecule comprising the desired polypeptide and an        N-terminal extension, said N-terminal extension being cleavable        from the desired polypeptide at a lysine cleavage site;    -   (ii) separating the expressed precursor molecule from the        culture broth;    -   (iii) preferentially acylating the ε-amino group of at least one        lysine in the desired polypeptide without acylating the ε-amino        group of the Lys-cleavage site in the N-terminal extension;    -   (iv) removing the N-terminal extension from the acylated        precursor molecule by enzymatic cleavage and    -   (v) isolating the acylated polypeptide by suitable means.

DETAILED DESCRIPTION OF THE INVENTION

The N-terminal extension will typically be up to 15 amino acids inlength and may be from 3-15; 3-12; 3-10; 3-9; 3-8; 3-7; 3-6; or 3-5amino acids in length. The amino acids in the N-terminal extension areselected with a multiple purpose: 1) to prevent or minimize acylation ofthe Lys cleavage site at the C-terminal end of the N-terminal extension;2) to protect the expressed precursor molecule against endoproteolyticdegradation; and 3) to prevent precipitation caused by fibrillationduring fermentation and down stream processing steps such as separationand purification in large scale production. Furthermore, the amino acidresidues at both ends of the N-terminal extension should be selected soas to ensure efficient cleavage of the N-terminal extension from thedesired polypeptide at the C-terminal end (at the Lys-cleavage site) andin the yeast cell at the N-terminal end from possible upstream sequencessuch as pre- or pre-pro peptides which have the purpose of ensuringtransport of the expressed polypeptide out of the host cell and into theculture medium. Finally, the N-terminal extension may serve as a tag forpurification purposes.

In one embodiment the present invention is related to a method, whereinone or more amino acid residues in the N-terminal extension are capableof establishing a metal ion complex binding site together with one ormore amino acid residues in the N-terminal end of the polypeptide.

The N-terminal extension comprising a metal ion binding site may bederived from the N-terminal of albumins. Examples of Cu⁺² binding sitesare the N-terminals of bovine (Asp-Thr-His-Lys, SEQ ID NO:34) and human(Asp-Ala-His-Lys, SEQ ID NO:35) serum albumin (Peters, T.; All aboutAlbumin, Academic Press, USA (1996) p 121-123) which could be used asthe N-terminal extension in synergy with heavy metal ions such as Cu⁺⁺present in the culture medium and during the acylation step (iii).

The N-terminal extension comprising a metal ion binding site may also bederived from the Zn-binding sites in especially some of themetalloendopeptidases EC 3.4.24-.

In one embodiment, the N-terminal extension comprises at least onehistidine residue.

The histidine residues in the N-terminal extension are typicallypositioned 1-4 residues from the lysine cleavage site.

Examples of N-terminal extensions comprising at least one histidineresidue are Glu-Glu-Ala-His-Lys(SEQ ID NO:1); Glu-(Glu-Ala)₂-His-Lys(SEQID NO:2); Glu-(Glu-Ala)₃His-Lys(SEQ ID NO:3); Glu-Glu-Gly-His-Lys(SEQ IDNO:4); Glu-His-Pro-Lys(SEQ ID NO:5); Glu-Glu-Gly-Glu-Pro-Lys(SEQ IDNO:6); Glu-Glu-His-Cys-Lys(SEQ ID NO:7); Glu-Glu-His-His-Lys(SEQ IDNO:8); Glu-His-His-His-Lys(SEQ ID NO:9); Glu-His-Ala-His-Lys(SEQ IDNO:10); Glu-Gly-Ala-His-Lys(SEQ ID NO:11); Glu-His-Gly-His-Gly-Lys(SEQID NO:12); Glu-Glu-Ala-His-Glu-Leu-Lys(SEQ ID NO:13);Glu-Glu-Ala-His-Glu-Ile-Lys(SEQ ID NO:14);Glu-Glu-Ala-His-Glu-Val-Lys(SEQ ID NO:15);Glu-Glu-Ala-His-Glu-Met-Lys(SEQ ID NO:16);Glu-Glu-Ala-His-Glu-Phe-Lys(SEQ ID NO:17);Glu-Glu-Ala-His-Glu-Tyr-Lys(SEQ ID NO:18);Glu-Glu-Ala-His-Glu-Trp-Lys(SEQ ID NO:19); Gln-Asp-Ala-His-Lys(SEQ IDNO:24); Glu-Glu-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:30);Asp-Thr-His-Lys(SEQ ID NO:34); Glu-His-His-Gly-His-Gly-Lys(SEQ IDNO:36); Asp-Ser-His-Lys(SEQ ID NO:37); Gln-Asp-Thr-His-Lys(SEQ IDNO:38); Glu-Ala-Glu-Ala-Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:39);Glu-Ala-Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:40);Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:41); Trp-His-Trp-Leu-Lys(SEQ IDNO:42); Glu-Glu-Trp-His-Trp-Leu-Lys(SEQ ID NO:43);Glu-Glu-Glu-Ala-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:44);Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:47);Glu-Ala-Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:48); andGlu-Ala-Glu-Ala-Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:49).

In another embodiment the present invention is related to a method,wherein the N-terminal extension of the precursor molecule comprises atleast one amino acid residue that is capable of establishing a saltbridge (ion bond) with the lysine cleavage site N-terminal to thepolypeptide.

In this embodiment, the N-terminal extension will typically comprise atleast one Glu or Asp which may be positioned between 1 to 5 residuesfrom the lysine cleavage site. In one such embodiment, the N-terminalextension comprises a Glu-Glu-sequence. Examples of N-terminalextensions comprising a Glu residue are Glu-Glu-Ala-Glu-Lys(SEQ IDNO:45); Glu-Glu-Gly-Glu-Pro-Lys(SEQ ID NO:46) and Glu-Glu-Lys. In thisembodiment, the N-terminal extension may furthermore be capable offorming an α-helix.

N-terminal extensions functioning both as a tag and being capable offorming a salt bridge are sequences derived from the enterokinase sitein bovine trypsinogen that is known to bind Ca⁺². Thus a suitableN-terminal extension for use in the present process isAsp-Asp-Asp-Asp-Lys(SEQ ID NO:26).

An N-terminal extension which is capable of forming an α-helix willtypically comprise a sequence that contains alternating polar andnon-polar amino acids as described by Zhu et al, Protein Science 2,384-394 (1993). Examples of such sequences areGlu-Glu-Ala-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:29).Glu-Glu-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:30);Leu-Asp-Gly-Arg-Leu-Glu-Ala-Leu-Lys(SEQ ID NO:31);Glu-Glu-Leu-Asp-Gly-Arg-Leu-Glu-Ala-Leu-Lys(SEQ ID NO:32);Glu-Glu-Leu-Asp-Ala-Arg-Leu-Glu-Ala-Leu-Lys(SEQ ID NO:33);Glu-Glu-Trp-His-Trp-Leu-Lys(SEQ ID NO:43); andGlu-Glu-Glu-Ala-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:44).

In this embodiment, the N-terminal extension may also comprise aeukaryotic N-glycosylation site N—X—S or N—X-T where X can be any aminoacid residue except Pro. Examples of N-terminal extensions comprising aglycosylation site are Glu-Glu-Gly-Asn-Thr-Thr-Pro-Lys(SEQ ID NO:20),Glu-Glu-Gly-Asn-Glu-Thr-Glu-Pro-Lys(SEQ ID NO:21),Glu-Glu-Gly-Asn-Asp-Thr-Glu-Pro-Lys(SEQ ID NO:22) andGlu-Glu-Gly-Asn-Thr-Thr-Glu-Pro-Lys (SEQ ID NO: 23).

In a further embodiment the present invention is related to a method,wherein the desired polypeptide is sensitive to proteolytic degradationat its N-terminal end and wherein the N-terminal extension prevents orminimizes such proteolytic degradation. Such polypeptides may becharacterized by having an Ala, Ser, Pro or Gly residue as the secondamino acid residue from the N-terminal end.

In a still further embodiment the polypeptide has a His or Tyr as theN-terminal amino acid residue.

In a more specific embodiment, the N-terminal extension has the formula

X_(n) - - - X₁-Lys

wherein Lys is a cleavage site and X_(n) - - - X₁ is a peptide sequenceof from 2-14 amino acid residues in length having the function ofpreventing or minimizing that the free ε-amino group in the Lys cleavagesite will be acylated in the above described step (iii). X_(n) - - - X₁will furthermore protect the expressed desired polypeptide fromendoproteolytic cleavage and will prevent precipitation caused byfibrillation of the N-terminal extended molecule during fermentation anddown stream separation and purification steps. The amino acid residuesin X_(n) - - - X₁ are furthermore selected so that optimal cleavage ofthe N-terminal extension at its C-terminal end (at Lys) is achieved.Furthermore, the amino acid residues in the N-terminal end of theextension are chosen so that cleavage is optimized in the yeast cellfrom upstream signal-leader-sequences at a KEX cleavage site (Lys, Arg).The amino acid residues in X_(n) - - - X₁ may in principle be any aminoacid residue except Lys as long as the peptide sequence fulfils at leastone of the required purposes with the proviso that at least one X is Hisor Glu or Asp and that number two amino acid residue from the N-terminalend of the extension is preferably not Ala or Pro.

In one embodiment X_(n) - - - X₁ is of 2-12 amino acid residues inlength. In another embodiment X_(n) - - - X₁ is of 2-10; 2-9; 2-8; 2-7;2-6; or 2-5 amino acid residues in length.

In another embodiment X_(n) - - - X₁ contains 2-8 amino acid residueswhich are selected from the group consisting of His; Glu; Ala; Asp; Gly;and Pro.

In another embodiment X_(n) - - - X₁ contains 4-10 amino acid residueswhich are selected from the group consisting of Glu; Asp; Ala; His; Trp;Tyr; Ile; Val; Met; and Phe.

In a further embodiment X_(n) - - - X₁ contains 5-8 amino acid residuesselected from the group consisting of Glu; Asp; Gly; Asn; Thr; Ser; andPro.

In all embodiments Glu and/or Asp are preferably selected as the firstand second amino acid residue from the N-terminal end of the extensionto ensure proper cleavage at this end from an upstream pre- orpre-pro-sequence by means of a Kex2p cleavage site. Furthermore, one ormore Glu and/Asp residues at the N-terminal end of the N-terminalextension will protect the N-terminal end of the ultimately desiredpolypeptide from in vivo degradation during fermentation.

Examples of X_(n) - - - X₁-Lys sequences are Glu-Glu-Ala-His-Lys(SEQ IDNO:1); Glu-(Glu-Ala)₂-His-Lys(SEQ ID NO:2); Glu-(Glu-Ala)₃His-Lys(SEQ IDNO:3); Glu-Glu-Gly-His-Lys(SEQ ID NO:4); Glu-His-Pro-Lys(SEQ ID NO:5);Glu-Glu-Gly-Glu-Pro-Lys(SEQ ID NO:6); Glu-Glu-His-Cys-Lys(SEQ ID NO:7);Glu-Glu-His-His-Lys(SEQ ID NO:8); Glu-His-His-His-Lys(SEQ ID NO:9);Glu-His-Ala-His-Lys(SEQ ID NO:10); Glu-Gly-Ala-His-Lys(SEQ ID NO:11);Glu-His-Gly-His-Gly-Lys(SEQ ID NO:12); Glu-Glu-Ala-His-Glu-Leu-Lys(SEQID NO:13); Glu-Glu-Ala-His-Glu-Ile-Lys(SEQ ID NO:14);Glu-Glu-Ala-His-Glu-Val-Lys(SEQ ID NO:15);Glu-Glu-Ala-His-Glu-Met-Lys(SEQ ID NO:16);Glu-Glu-Ala-His-Glu-Phe-Lys(SEQ ID NO:17);Glu-Glu-Ala-His-Glu-Tyr-Lys(SEQ ID NO:18);Glu-Glu-Ala-His-Glu-Trp-Lys(SEQ ID NO:19);Glu-Glu-Gly-Asn-Thr-Thr-Pro-Lys(SEQ ID NO:20);Glu-Glu-Gly-Asn-Glu-Thr-Glu-Pro-Lys(SEQ ID NO:21),Glu-Glu-Gly-Asn-Asp-Thr-Glu-Pro-Lys(SEQ ID NO:22);Glu-Glu-Gly-Asn-Thr-Thr-Glu-Pro-Lys(SEQ ID NO: 23);Gln-Asp-Ala-His-Lys(SEQ ID NO:24); Glu-Glu-Lys; Asp-Asp-Asp-Asp-Lys(SEQID NO:26); Glu-Glu-Ala-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:29);Glu-Glu-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:30);Leu-Asp-Gly-Arg-Leu-Glu-Ala-Leu-Lys(SEQ ID NO:31);Glu-Glu-Leu-Asp-Gly-Arg-Leu-Glu-Ala-Leu-Lys (SEQ ID NO:32);Glu-Glu-Leu-Asp-Ala-Arg-Leu-Glu-Ala-Leu-Lys(SEQ ID NO:33);Asp-Thr-His-Lys(SEQ ID NO:34); Asp-Ala-His-Lys(SEQ ID NO:35);Glu-His-His-Gly-His-Gly-Lys(SEQ ID NO:36); Asp-Ser-His-Lys(SEQ IDNO:37); Gln-Asp-Thr-His-Lys(SEQ ID NO:38);Glu-Ala-Glu-Ala-Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:39);Glu-Ala-Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:40);Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:41); Trp-His-Trp-Leu-Lys(SEQ IDNO:42); Glu-Glu-Trp-His-Trp-Leu-Lys(SEQ ID NO:43);Glu-Glu-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:44);Glu-Glu-Ala-Glu-Lys(SEQ ID NO:45); Glu-Glu-Gly-Glu-Pro-Lys(SEQ IDNO:46); Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:47);Glu-Ala-Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:48);Glu-Ala-Glu-Ala-Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:49);

In a further embodiment, the N-terminal extension has the sequenceAsp-X-His-Lys(SEQ ID NO:50) where X is Ala, Thr or Ser. The sequenceAsp-X-His-Lys constitutes a heavy metal ion albumin binding site.

In a still further embodiment, the N-terminal extension has the sequenceHis-Z₁—Z₂-Lys(SEQ ID NO:51) wherein Z₁ is Glu; Asp; Asn; Gln; Ser; Thr;Gly; Leu; Ile; Val; Met; Phe or Tyr and Z₂ is Leu; Ile; Val; Met; Phe;Tyr; Trp or Cys. In one embodiment Z₁ is Glu or Asp. The sequenceHis-Z₁—Z₂-Lys will together with an N-terminal His residue in thedesired polypeptide constitute a metal binding site homologue toZn-binding sites in certain metalloendopeptidases such asGlu-Glu-Ala-His-Glu-Leu-Lys(SEQ ID NO:13);Glu-Glu-Ala-His-Glu-Ile-Lys(SEQ ID NO:14);Glu-Glu-Ala-His-Glu-Val-Lys(SEQ ID NO:15);Glu-Glu-Ala-His-Glu-Met-Lys(SEQ ID NO:16);Glu-Glu-Ala-His-Glu-Phe-Lys(SEQ ID NO:17);Glu-Glu-Ala-His-Glu-Tyr-Lys(SEQ ID NO:18); andGlu-Glu-Ala-His-Glu-Trp-Lys(SEQ ID NO:19).

The N-terminal extension is found to be stably attached to the precursormolecule of the invention during fermentation, protecting the N-terminalend of the precursor molecule against the proteolytic activity of yeastproteases such as Ste13p or Dap2p.

The N-terminal extension will be removed from the acylated recoveredprecursor molecule by means of a proteolytic enzyme which is specificfor Lys. Examples of such proteolytic enzymes are trypsin orAchromobacter lyticus protease 1.

According to a further aspect the present invention is related to apolypeptide precursor for a desired polypeptide said polypeptideprecursor having the formula

N-terminal-extension-Lys-Z₃—Z₄-*polypeptide*

-   -   wherein Lys is a cleavage site, the N-terminal extension has        2-14 amino acid residues as described above, Z₃ is the        N-terminal amino acid residue in the desired polypeptide and is        His or Tyr, Z₄ is the next amino acid residue from the        N-terminal end in the desired polypeptide and is Ala, Ser or        Gly, and *polypeptide* is the remaining sequence of the desired        polypeptide.

In one embodiment of the present invention *polypeptide* is the relevantportion of GLP-1 or GLP-2.

Introduction of lipophilic acyl groups in naturally occurring peptidesor analogues thereof has shown to lead to acylated peptides which have aprotracted profile relative to the native peptide or unmodifiedanalogues. This phenomenon is disclosed and demonstrated in WO 98/08871which discloses acylation of GLP-1 and analogues thereof and in WO98/08872 which discloses acylation of GLP-2 and analogues thereof. Thelipophilic group may be introduced by means of mono- or dipeptidespacers as disclosed in WO 98/08871. Alternatively, the lipophilic groupmay be introduced by means of α-amino-α,ω-dicarboxylic acid groups asdisclosed in WO 00/55119.

The present polypeptide precursor molecules will contain at least twolysine groups with a free ε-amino group, i.e. the lysine cleavageresidue and at least one lysine residue in the desired polypeptide. Ifall lysine groups were acylated including the lysine cleavage residue,subsequent cleavage of the N-terminal extension from the desiredpolypeptide sequence will not be possible. Thus one of the purposes toexpress the desired polypeptide with an N-terminal extension is toprevent or minimize acylation of the free ε-amino group in the lysinecleavage site. The precursor molecule can then be preferentiallyacylated in the desired lysine residue which in the case of GLP-1 is thelysine in position 26. After acylation the acylated precursor moleculeis cleaved by suitable enzymatic means as described above and thedesired acylated polypeptide can be isolated.

The acylation step (iii) may be conducted at a pH between 7 and 12. Incertain embodiments, the pH will be between 8 and 11.5 or between 9.0and 10.5 and a pH value of about 9.5 to 10.5 has proven to be efficient.The temperature will be between minus 5 and 35° C. and will typically bebetween 0 and 20° C. or between 15 and 30° C.

In one embodiment of the present invention the acylation is conducted inthe presence of divalent metal ions such as Cu²⁺, Fe²⁺, Ni²⁺, Co²⁺,Zn²⁺, Mg²⁺, Mn²⁺ and Ca²⁺. However, trivalent metal ions such as Co³⁺are also efficient for the purpose of the present invention.

According to further aspects the present invention is related topolynucleotides encoding the claimed polypeptide precursors and vectorsand transformed host cells containing such polynucleotides.

DEFINITIONS

The term “preferential acylating” is meant to include and acylationprocess where acylation takes place at one or more preferred positionsin the molecule in a substantial higher degree than at other positionsin the same molecule. Thus, the acylation at the preferred positions isat least 50%, preferably at least 80% and most preferred 90-100% of thetotal acylation. In the present method acylation of the ε-amino group ofthe lysine cleavage site in the N-terminal extension should be avoidedor minimized as much as possible as acylation at this position mayinterfere with the subsequent cleavage of the N-terminal extension fromthe desired end product leading to yield loss.

With “N-terminal extension” is meant a polypeptide sequence removablyattached to the N-terminal amino acid residue in the desiredpolypeptide. The N-terminal extension may be 2-15 amino acid residues inlength and will comprise a Lys residue as its C-terminal amino acidresidue for cleavage from the desired polypeptide. The N-terminalextension will protect the expressed fusion polypeptide againstproteolytic degradation within the host cell as described above. Inaddition, it is believed to prevent or minimize acylation of the ε-aminogroup of the lysine cleavage site in the N-terminal extension by maskingsaid Lys residue during the acylation process.

With “desired polypeptide” is meant the ultimate polypeptide obtainedafter cleavage of the N-terminal extension from the precursor molecule.This expression will cover both the acylated and non-acylated version ofsaid polypeptide. The “N-terminal extension” includes the lysine residuewhich constitutes the cleavage site for cleavage of the N-terminalextension from the desired polypeptide's N-terminal end. It will beunderstood that whenever a Lys-group is shown as the C-terminal aminoacid of an N-terminal extension or a sequence being comprised in theN-terminal extension, then said Lys-residue is directly linked to theN-terminal amino acid residue of the desired polypeptide and constitutesthe cleavage site. Cleavage at the Lys-residue (step iv) is preferablyaccomplished by means of a proteolytic enzyme which is specific Lys.Examples of such proteolytic enzymes are trypsin or Achromobacterlyticus protease (ALP).

An example of a desired polypeptide is GLP-1. The amino acid sequence ofGLP-1 is given i.a. by Schmidt et al. (Diabetologia 28 704-707 (1985).Although the interesting pharmacological properties of GLP-1(7-37) andanalogues thereof have attracted much attention in recent years onlylittle is known about the structure of these molecules. The secondarystructure of GLP-1 in micelles has been described by Thorton et al.(Biochemistry 33 3532-3539 (1994)), but in normal solution, GLP-1 isconsidered a very flexible molecule.

A simple system is used to describe fragments and analogues of thispeptide. Thus, for example, Gly⁸GLP-1₍₇₋₃₇₎ designates a fragment ofGLP-1 derived from GLP-1₍₁₋₃₇₎ by deleting the amino acid residues Nos.1 to 6 and substituting the naturally occurring amino acid residue inposition 8 (Ala) by Gly. Similarly,Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1₍₇₋₃₇₎ designates GLP-1₍₇₋₃₇₎ whereinthe ε-amino group of the Lys residue in position 26 has beentetradecanoylated.

Other examples of a desired polypeptides are GLP-2 and glucagon bothbelonging to the GRF (growth hormone releasing factor) family ofpeptides having a His or Tyr in the N-terminal position and Ser, Ala orGly in the next position, vide Adelhorst K. et al., The Journal ofBiological Chemistry (1994) p 6275-6278).

“POT” is the Schizosaccharomyces pombe triose phosphate isomerase gene,and “TPI1” is the S. cerevisiae triose phosphate isomerase gene.

With “fibrillation” is meant a process where so called “fibrils” areformed. “Fibrils” is a well recognized and described phenomenon and maybe composed of antiparallel β-sheets. Molecules like GLP's with littleα-helical structure and a very flexible and little tertiary structureare very prone to aggregation that leads to precipitation and loss ofyield if very crude chemical conditions are not taken in use such asalkaline treatment at pH ˜12.

By a “leader” is meant an amino acid sequence consisting of apre-peptide (the signal peptide) and a pro-peptide.

The term “signal peptide” is understood to mean a pre-peptide which ispresent as an N-terminal sequence on the precursor form of a protein.The function of the signal peptide is to allow the heterologous proteinto facilitate translocation into the endoplasmic reticulum.

The signal peptide is normally cleaved off in the course of thisprocess. The signal peptide may be heterologous or homologous to theyeast organism producing the protein. A number of signal peptides may beused with the DNA construct of the invention including the YPS1 signalpeptide (formally called the YAP3 signal peptide) or any functionalanalogue thereof (Egel-Mitani et al. (1990) YEAST 6:127-137 and U.S.Pat. No. 5,726,038) and the α-factor signal of the MFα1 gene (Thorner(1981) in The Molecular Biology of the Yeast Saccharomyces cerevisiae,Strathern et al., eds., pp 143-180, Cold Spring Harbor Laboratory, NYand U.S. Pat. No. 4,870,00.

The term “pro-peptide” means a polypeptide sequence whose function is toallow the expressed polypeptide to be directed from the endoplasmicreticulum to the Golgi apparatus and further to a secretory vesicle forsecretion into the culture medium (i.e. exportation of the polypeptideacross the cell wall or at least through the cellular membrane into theperiplasmic space of the yeast cell). The pro-peptide may be the yeastα-factor pro-peptide, vide U.S. Pat. Nos. 4,546,082 and 4,870,008.Alternatively, the pro-peptide may be a synthetic pro-peptide, which isto say a pro-peptide not found in nature. Suitable syntheticpro-peptides are those disclosed in U.S. Pat. Nos. 5,395,922; 5,795,746;5,162,498 and WO 98/32867. The pro- peptide will preferably contain anendopeptidase processing site at the C-terminal end, such as a Lys-Argsequence or any functional analog thereof.

The polynucleotide sequence of the invention may be preparedsynthetically by established standard methods, e.g. the phosphoamiditemethod described by Beaucage et al. (1981) Tetrahedron Letters22:1859-1869, or the method described by Matthes et al. (1984) EMBOJournal 3:801-805. According to the phosphoamidite method,oligonucleotides are synthesized, for example, in an automatic DNAsynthesizer, purified, duplexed and ligated to form the synthetic DNAconstruct. A currently preferred way of preparing the DNA construct isby polymerase chain reaction (PCR).

The polynucleotide sequence of the invention may also be of mixedgenomic, cDNA, and synthetic origin. For example, a genomic or cDNAsequence encoding a leader peptide may be joined to a genomic or cDNAsequence encoding the precursor molecule of the invention, after whichthe DNA sequence may be modified at a site by inserting syntheticoligonucleotides encoding the desired amino acid sequence for homologousrecombination in accordance with well-known procedures or preferablygenerating the desired sequence by PCR using suitable oligonucleotides.

The invention encompasses a vector which is capable of replicating inthe selected microorganism or host cell and which carries apolynucleotide sequence encoding the precursor molecule of theinvention. The recombinant vector may be an autonomously replicatingvector, i.e., a vector which exists as an extra-chromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, an extra-chromosomal element, a mini-chromosome, or anartificial chromosome. The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon may beused. The vector may be linear or closed circular plasmids and willpreferably contain an element(s) that permits stable integration of thevector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

In a preferred embodiment, the recombinant expression vector is capableof replicating in yeast. Examples of sequences which enable the vectorto replicate in yeast are the yeast plasmid 2 μm replication genes REP1-3 and origin of replication.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Selectable markers for usein a filamentous fungal host cell include amdS (acetamidase), argB(ornithine carbamoyltransferase), pyrG (orotidine-5′-phosphatedecarboxylase) and trpC (anthranilate synthase). Suitable markers foryeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Apreferred selectable marker for yeast is the Schizosaccharomyces pompeTPI gene (Russell (1985) Gene 40:125-130).

In the vector, the polynucleotide sequence is operably connected to asuitable promoter sequence. The promoter may be any nucleic acidsequence which shows transcriptional activity in the host cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extra-cellular or intra-cellular polypeptides eitherhomologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacilluslicheniformis penicillinase gene (penP). Examples of suitable promotersfor directing the transcription in a filamentous fungal host cell arepromoters obtained from the genes for Aspergillus oryzae TAKA amylase,Rhizomucor miehei aspartic proteinase, Aspergillus niger neutralalpha-amylase, and Aspergillus niger acid stable alpha-amylase. In ayeast host, useful promoters are the Saccharomyces cerevisiae MFα1, TPI,ADHm Gal or PGK promoters.

The polynucleotide construct of the invention will also typically beoperably connected to a suitable terminator. In yeast a suitableterminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl.Genet. 1:419-434) or the CYC1 terminator.

The procedures used to ligate the polynucleotide sequence of theinvention, the promoter and the terminator, respectively, and to insertthem into a suitable vector containing the information necessary forreplication in the selected host, are well known to persons skilled inthe art. It will be understood that the vector may be constructed eitherby first preparing a DNA construct containing the entire DNA sequenceencoding the precursor molecule of the invention, and subsequentlyinserting this fragment into a suitable expression vector, or bysequentially inserting DNA fragments containing genetic information forthe individual elements followed by ligation.

The present invention also relates to recombinant host cells, comprisinga polynucleotide sequence encoding the precursor molecule of theinvention. A vector comprising such polynucleotide sequence isintroduced into the host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The host cell may be a prokaryote or aeukaryote cell. Useful prokaryotes are bacterial cells such as grampositive bacteria including Bacillus and Streptomyces cells, or gramnegative bacteria such as E. coli and Pseudomonas sp. Cells. Eukaryotecells may be mammalian, insect, plant, or fungal cells. In a oneembodiment, the host cell is a yeast cell. The yeast organism used inthe process of the invention may be any suitable yeast organism which,on cultivation, produces large amounts of the precursor molecule.Examples of suitable yeast organisms are strains selected from the yeastspecies Saccharomyces cerevisiae, Saccharomyces kluyveri,Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis,Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichiakluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candidacacaoi, Geotrichum sp., and Geotrichum fermentans.

The transformation of the yeast cells may for instance be effected byprotoplast formation followed by transformation in a manner known perse. The medium used to cultivate the cells may be any conventionalmedium suitable for growing yeast organisms. The secreted precursor ofthe invention may then be recovered from the medium by conventionalprocedures including separating the yeast cells from the medium bycentrifugation, filtration or catching the precursor by an ion exchangematrix or by a reverse phase absorption matrix, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulphate, followed by purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography, affinitychromatography, or the like.

In the present text, the designation “an analogue” is used to designatea peptide wherein one or more amino acid residues of the parent peptidehave been substituted by another amino acid residue and/or wherein oneor more amino acid residues of the parent peptide have been deletedand/or wherein one or more amino acid residues have been added to theparent peptide. Such addition can take place either at the N-terminalend or at the C-terminal end of the parent peptide or both.

The term “derivative” is used in the present text to designate a peptidein which one or more of the amino acid residues of the parent peptidehave been chemically modified, e.g. by alkylation, acylation, esterformation or amide formation.

The amino acid residues are either indicated by the one letter or thethree letter code.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the plasmid pKV304 which contain DNA encodingArg³⁴GLP-1₍₇₋₃₇₎ under regulatory control of the TPI promoter and-terminator and the MFalpha prepro sequence. This plasmid is thestarting plasmid for making expression plasmids for the precursormolecules according to the present invention.

EXAMPLES Example 1 Expression of N-Terminally Extended Arg³⁴GLP-1₍₇₋₃₇₎

The host strain ME1719 is a diploid strain and has a phenotype whichlacks two aspartyl protease activities, i.e. YPS1 (previously calledYAP3) which cleaves C-terminal side of mono- or dibasic amino acidresidues (Egel-Mitani, et al., YEAST 6: 127-137, 1990) and PEP4 avacuolar protease A responsible for activation of other proteases suchas protease B, carboxypeptidase Y, aminopeptidase I, RNase, alkalinephosphatase, acid threhalase and exopolyphosphatase. Moreover the triosephosphate isomerase gene (TPI) has been disrupted which phenotype makesit possible to utilize glucose in transformants grown on glucosecontaining medium. The genetic background of ME1719 isMATa/αΔyps1::ura3/Δyps1::URA3 pep4-3/pep4-3 Δtpi::LEU2/Δtpi::LEU2leu2/leu2 Δura3/Δura3.

Expression plasmids containing the N-terminally extendedArg³⁴GLP-1₍₇₋₃₇₎ were made as follows: Plasmid pKV304 containing DNAencoding Arg³⁴GLP-1₍₇₋₃₇₎ without an N-terminal extension was digestedwith either Eagl+Ncol or Eagl+Asp718. After agarose electrophoresis andGeneClean™ III purification, fragments of 1.4 kb and 10 kb, respectivelywere isolated. Oligonucleotide adaptors corresponding to variousN-terminal extensions of Arg³⁴GLP-1₍₇₋₃₇₎ containing Ncol and Asp718cleavage sites were likewise purified as described above. The 1.4 kbfragment (Eagl+Ncol), 10 kb fragment (Eagl+Asp718) and the adaptorfragment designed for the N-terminal extension of Arg³⁴GLP-1₍₇₋₃₇₎(Ncol+Asp718) were ligated and transformed in E. coli strain MT172 andplasmid DNA was sequenced to verify the correct N-terminally extendedArg³⁴GLP-1₍₇₋₃₇₎.

Plasmid DNA was then transformed into yeast strain ME1719 and yeasttransformants were isolated twice on MUPD selective plates. Yeast cellswere cultured in 5 ml MUPD medium for 3 days at 30° C. and culturesupernatants were analyzed by HPLC and MALDI-MS (Matrix Assisted LaserDesorption/Inonisation Mass Spectrometry). MUPD medium consists of 25 gyeast extract (Bacto), 5 g KH₂PO₄, 1.5 g MgSO₄.7H₂O, ion exchanged waterto 1 liter and pH adjusted to 6.0 with 5 N H₂SO₄ before autoclavation at121° C., 20 min. 30 g glucose was separately autoclaved in a 50% w/vconcentration and added aseptically.

Table 1 shows the different GLP-1 precursors and the yield compared to acontrol with no N-terminal extension.

TABLE 1 Yield % of con- Extension Polypeptide trol None Arg³⁴GLP-1₍₇₋₃₇₎100 (control) EEK Arg³⁴GLP-1₍₇₋₃₇₎ 206 EEAEK (SEQ ID NO: 45)Arg³⁴GLP-1₍₇₋₃₇₎ 217 HK Arg³⁴GLP-1₍₇₋₃₇₎  63 E(EA)HK (SEQ ID NO: 1)Arg³⁴GLP-1₍₇₋₃₇₎ 233 E(EA)₂HK (SEQ ID NO: 2) Arg³⁴GLP-1₍₇₋₃₇₎ 208E(EA)₃HK (SEQ ID NO: 3) Arg³⁴GLP-1₍₇₋₃₇₎ 223 EEGHK (SEQ ID NO: 4)Arg³⁴GLP-1₍₇₋₃₇₎ 171 EHPK (SEQ ID NO: 5) Arg³⁴GLP-1₍₇₋₃₇₎ 132 EEGEPK(SEQ ID NO: 6) Arg³⁴GLP-1₍₇₋₃₇₎ 211 EEHCK (SEQ ID NO: 7)Arg³⁴GLP-1₍₇₋₃₇₎  58 EEHHK (SEQ ID NO: 8) Arg³⁴GLP-1₍₇₋₃₇₎ 125 EHHHK(SEQ ID NO: 9) Arg³⁴GLP-1₍₇₋₃₇₎  72 EHAHK (SEQ ID NO: 10)Arg³⁴GLP-1₍₇₋₃₇₎  76 EGAHK (SEQ ID NO: 11) Arg³⁴GLP-1₍₇₋₃₇₎  97 EHGHGK(SEQ ID NO: 12) Arg³⁴GLP-1₍₇₋₃₇₎  73 EEAHELK (SEQ ID NO: 13)Arg³⁴GLP-1₍₇₋₃₇₎ 220 EEAHEIK (SEQ ID NO: 14) Arg³⁴GLP-1₍₇₋₃₇₎ 128EEAHEVK (SEQ ID NO: 15) Arg³⁴GLP-1₍₇₋₃₇₎ 217 EEAHEMK (SEQ ID NO: 16)Arg³⁴GLP-1₍₇₋₃₇₎ 232 EEAHEFK (SEQ ID NO: 17) Arg³⁴GLP-1₍₇₋₃₇₎ 226EEAHEYK (SEQ ID NO: 18) Arg³⁴GLP-1₍₇₋₃₇₎ 200 EEAHEWK (SEQ ID NO: 19)Arg³⁴GLP-1₍₇₋₃₇₎ 200 EEGNTTPK (SEQ ID NO: 20) Arg³⁴GLP-1₍₇₋₃₇₎ 216EEGNETEPK (SEQ ID NO: 21) Arg³⁴GLP-1₍₇₋₃₇₎ 145 EEGNDTEPK (SEQ ID NO: 22)Arg³⁴GLP-1₍₇₋₃₇₎ 175 EEGNTTEPK (SEQ ID NO: 23) Arg³⁴GLP-1₍₇₋₃₇₎  31*QDAHK (SEQ ID NO: 24) Arg³⁴GLP-1₍₇₋₃₇₎  55 QDTAK (SEQ ID NO: 25)Arg³⁴GLP-1₍₇₋₃₇₎  66 DDDDK (SEQ ID NO: 26) Arg³⁴GLP-1₍₇₋₃₇₎ 190EAEAWHWLK (SEQ ID NO: 27) Arg³⁴GLP-1₍₇₋₃₇₎  36 EAEAEAWHWLK (SEQ ID NO:28) Arg³⁴GLP-1₍₇₋₃₇₎  34 EEAEAWHWLK (SEQ ID NO: 29) Arg³⁴GLP-1₍₇₋₃₇₎  20EEEAWHWLK (SEQ ID NO: 30) Arg³⁴GLP-1₍₇₋₃₇₎ n.d. LDGRLEALK (SEQ ID NO:31) Arg³⁴GLP-1₍₇₋₃₇₎  58 EELDGRLEALK (SEQ ID NO: 32) Arg³⁴GLP-1₍₇₋₃₇₎211 EELDARLEALK (SEQ ID NO: 33) Arg³⁴GLP-1₍₇₋₃₇₎ 239 *Product mainlyhyperglycosylated

Example 2 Acylation of EEAHK(SEQ ID NO:1)-Arg³⁴GLP-1₍₇₋₃₇₎ in thePresence and Absence of Divalent or Trivalent Metal Ions

The GLP-1 analogue was produced as described in Example 1. Thefermentation broth was clarified by centrifugation and 2620 ml ofsupernatant was diluted to 7900 ml and pH was adjusted to pH 3.1. Thefinal conductivity was 4.9 mS/cm. A 2.6×100 cm column packed with 100 mlPharmacia Streamline® SP Code no. 17-0993-05 was equilibrated andfluidised as recommended by the supplier (Pharmacia booklet 18-1124-26,Expanded Gel Adsorption, Principles and Methods) employing a 0.025Mcitrate buffer pH 3.1 and subsequently eluted by 0.5M Tris base at aflow of 0.5 ml/min. The fractions containing the GLP1 analogue wasidentified by analytical RP-HPLC employing a gradient of CH₃CN in 0.010MTris, 0.015M Na₂SO₄ pH 7.4 with diluted H₂SO₄. The volume of the pooledsamples was 100 ml containing 361 mg of the GLP-1 analogue and thepurity was 72.4%. The sample was further purified by preparativeRP-HPLC. The buffer system consisted of an A-buffer 0.010M Tris, 0.015MNa₂SO₄ and 20% ethanol v/v pH 7.5 with diluted H₂SO₄ and an B-bufferconsisting of 70% ethanol. Aliquots corresponding to 90 mg GLP1 analoguewas applied to a HPLC column (250×20) mm packed with Nucleosil 300 Å, 7μm, C4 obtained from Macherey-Nagel, D, equilibrated with 10% B thesample was eluted with a linear gradient from 10% B to 90% B in a totalof 720 ml at a flow rate of 6 ml min. The eluent was monitored at 214 nmand 276 nm. The samples containing the GLP1 analogue were pooled,diluted with one volume of water, adjusted to pH 5.0 and cooled to 4° C.The precipitate was isolated by centrifugation and lyophilised. 286 mgwas obtained and the final purity was 98.0%.

Acylation of the GLP-1 analogue was performed employing 10 mg samples ofpurified analogue. The sample was dissolved in 0.5 ml 0.05M Na₂CO₃ andincubated at 15° C. Glu(ONSU)N-hexadecanoyl methylester was dissolved in0.5 ml CH₃CN and added to the analogue solution. Samples were takenbefore addition of reagent and after 15 and 30 minutes and aftertermination of the reaction with quenching buffer. The samples wereadded quenching buffer and diluted with 20% vol/vol ethanol and analysedby analytical RP-HPLC. Acylation in the presence of divalent metal ionswas conducted by addition of the appropriate volume of a 0.1M solutionof the metal ion before addition of the amino acid acylation reagent.The volume of Na₂CO₃ buffer was adjusted accordingly. Zn²⁺ acetate wasused in experiment with Zn²⁺ as the metal ion.

Optimization of Acylation of EEAHK(SEQ IDNO:1)-Arg³⁴GLP-1₍₇₋₃₇₎-Lys²⁶γ-Glu-hexadecanoyl in the Presence of Zn²⁺

Addition of more than 2 equivalents of Zn²⁺ was associated byprecipitation of the sample. However, the yield improved when 2equivalents of Zn²⁺ was used for acylation together with a surplus ofacylation reagent. Thus, a yield of 52% was obtained together with 7% ofdesamidated acylation product, which is superior to 42.4% yield and 5.1%desamidated product obtained in the absence of Zn²⁺.

The results are shown in Table 2 and Table 3 below.

TABLE 2 Acylation of EEAHK(SEQ ID NO: 1)-Arg³⁴GLP-1₍₇₋₃₇₎ at pH 10.2,50% CH₃CN by 1.3 equivalents Glu(ONSU)N-hexadecanoyl methylester 15 min30 min 30 min stop Reaction GLP-1 0 min GLP-1 product GLP-1 productGLP-1 product 0.0 equivalents Zn²⁺  98.% 36.2% 41.4% 35.5% 42.4%* 36.1%45.2 0.5 equivalents Zn²⁺ 98.3% 30.2% 42.9% 32.6% 41.7% 1.0 equivalentsZn²⁺ 99.5% 33.8% 38.6% 34.8% 34.6% 1.5 equivalents Zn²⁺ 99.3% 30.7%36.3% 29.2% 35.5% 31.7% 34.2% 2.0 equivalents Zn²⁺ 99.2% 28.1% 44.3%22.9% 46.8% 30.0% 42.8% 2.5 equivalents Zn²⁺ 99.3% 25.0% 39.8% 21.3%46.6% 22.5% 45.0% *% 5.1% of desamidated product was obtained in thissynthesis

TABLE 3 Acylation of EEAHK(SEQ ID NO: 1)-Arg³⁴GLP-1₍₇₋₃₇₎ at pH 10.2,50% CH₃CN by 2 and 3 equivalents Glu(ONSU)N-hexadecanoyl methylester and2 equivalents Zn²⁺ Reaction 15 min 30 min 30 min stop Acylation reagent0 min GLP-1 product GLP-1 product GLP-1 product 2 equivalents acylation98.5% 16.9% 37.8% 11.6% 39.0% 12.9% 38.4% reagent 3 equivalentsacylation 98.3% 6.0% 35.7% 3.8% 33.2% reagent 2 equivalents Zn²⁺ 98.127.8% 52.0% 16.4% 52.0%* 19.9% 46.1% 2 equivalents acylation reagent 2equivalents Zn²⁺ 98.6% 13.2% 36.6% 15.8% 38.4% 13.2% 41.6% 3 equivalentsacylation reagent *% 7.0% of desamidated product was obtained in thissynthesis.

Synthesis of Arg³⁴GLP-1₍₇₋₃₇₎-Lys²⁶γ-Glu-hexadecanoyl in the Presence ofZn²⁺

EEAHK(SEQ ID NO:1)-Arg³⁴GLP-1₍₇₋₃₇₎ was acylated by addition of 2equivalents of acylation reagent in the presence of 2 equivalents Zn²⁺for 15 min. as described above. A total of 15 μl was applied to ananalytical RP-HPLC column using a TFA/CH₃CN/H₂O buffer system. Thepurified product corresponded to 57 μg represent a synthesis yield of46%. The product was dried and subsequently dissolved in 0.025 ml 0.1 mNaOH at 0° C. and incubated for 20 min. After hydrolysis of themethylester, the sample was then added 15 μl 0.1 m HCl and 5 μl 0.1 mTris. HCl and a pH indicator paper measured a pH of approximately pH8-9. The sample was then added 10 μl CH₃CN and thereafter 1 μl 1 mg/mlAchromobacter lyticus protease I (EC 3.4.21.50) and incubated for 30 minat room temperature. A total of 10 μl was applied to the RP-HPLC systemas described above and the most prominent of the 3 peaks corresponding66.2% was further characterised and found to be identical to the desiredproduct. The results are shown in Table 4 below.

TABLE 4 MW Peak calculated MW found Identity Yield Peak 1 — No signalGLP-1 analogue 8.5% Peak 2 3749.29 3748.41 Arg³⁴ GLP-1₍₇₋₃₇₎-Lys²⁶-66.2% γ-Glu-hexadecanoyl Peak 3 3749.29 3761.8 Unknown derivative of19.6% GLP-1

Example 3 Acylation and ALP-Processing of EEAEK(SEQ ID NO:45)-Arg³⁴GLP-1₍₇₋₃₇₎

The GLP-1 precursor EEAEK(SEQ ID NO:43)-Arg³⁴GLP-1₍₇₋₃₇₎ was expressedin yeast as described above and recovered by adjusting the ionicstrength to 7 in the fermentation supernatant and binding to a cationiccolumn SP Sepharose Fast Flow XL (Amersham-Pharmacia). The precursor waseluted with a gradient 0-100% of 50 mM formic acid+1 M NaCl in 50 mMformic acid. After 10 column volumes the column was washed with 2volumes of 50 mM formic acid followed by elution with 0.5 M glycine pH 9for 3.5 column volumes. The fractions were analysed by MALDI-MS and theprecursor pools were identified. These pools were applied on ReversePhase HPLC a ZORBAX 300SB-CN column (9.4×250 mm). Elution was carriedout with a gradient of 30-70% of buffer B (0.1% TFA/80% ethanol) inbuffer A (0.1% TFA). Fractions were identified by UV-pattern andMALDI-MS. Pools were collected, the ethanol evaporated in vacuum and theproduct was lyophilised.

5 mg of lyophilised precursor EEAEK(SEQ ID NO:45)-Arg³⁴GLP-1₍₇₋₃₇₎ wasdissolved in 350 μl water and 700 μl NMP (N-methyl-pyrrolidon-2) at roomtemperature and 4 equivalents of N-hexadecanoyl-/-glutamicacid-α-tert-butyl ester-γ-succinylimidyl ester was added at time zero.The pH was adjusted to 9.5 with EDIPA (ethyl di-isopropyl amin) and keptconstant at 9.5 by addition of EDIPA. After 60 minutes the reaction wasstopped by addition of 208 μl of a solution of 1% glycin in water.Analysis of the reaction mixture showed that 24% was unreacted product.61% was monoacylated in position Lys²⁶. 8% was diacylated in Lys²⁶ andat the N-terminal amino group. 7% was diacylated in Lys⁶ and Lys²⁶. Themono- and diacylated molecules were converted to monoacylatedArg³⁴GLP-1₍₁₋₃₇₎ (69%) by processing at the lysine clevage site with ALPaccording to well established procedures. The result was confirmed byHPLC and MALDI-MS.

Example 4 Acylation and Cleavage of EELDARLEALK(SEQ IDNO:33)-Arg³⁴GLP-1₍₁₋₃₇₎, EEAHEYK(SEQ ID NO:18)-Arg³⁴GLP-1₍₁₋₃₇₎ andDDDDK(SEQ ID NO: 26)-Arg³⁴GLP-1₍₁₋₃₇₎

The GLP-1 precursors EELDARLEALK(SEQ ID NO:33)-Arg³⁴GLP-1₍₁₋₃₇₎,EEAHEYK(SEQ ID NO:18)-Arg³⁴GLP-1₍₁₋₃₇₎ and DDDDK(SEQ ID NO:26)-Arg³⁴GLP-1₍₁₋₃₇₎ were all expressed in yeast and purified asdescribed in Example 3.

Acylation was carried out as described in Example 3. 1.3 Equivalentswere used of hexadecanoyl-/-glutamic acid-α-tert-butylester-γ-succinylimidyl ester. HPLC and MALDI-MS analysis revealed in allcases ˜15-30% unreacted substrate and around 20% diacylated product(Lys⁶, Lys²⁶) after hydrolysis with ALP directly in the acylationmixture. The results are shown in table 5.

TABLE 5 Only monoacylated N-terminal extended Lys²⁶- GLP1 and diacylatedN-terminal ex- tended Lys⁶, Lys²⁶-GLP1 peaks determined % % Monoacy-Diacylated lated Lys⁶, Extension Lys²⁶ Lys^(26**) DDDDK (SEQ ID NO: 26)80 20 EELDARLEALK (SEQ ID NO: 33) 76 24 EEAHEYK (SEQ ID NO: 18) 76 24^(**)N-terminal extended (not cleaved)

1. A method for making an acylated polypeptide, wherein said polypeptidecomprises at least one lysine residue that is acylated on its ε-aminogroup, said method comprising: (i) expressing in a suitable host cell aprecursor of said polypeptide, wherein said precursor comprises saidpolypeptide and an N-terminal extension, said N-terminal extension beingcleavable from the polypeptide at a lysine cleavage site; (ii)preferentially acylating the ε-amino group of said at least one lysineresidue in the polypeptide without acylating the ε-amino group of thelysine cleavage site, to produce an acylated precursor; and (ii)removing the N-terminal extension from the acylated precursor byenzymatic cleavage to produce said acylated polypeptide.
 2. A methodaccording to claim 1, wherein the N-terminal extension is up to 15 aminoacids in length.
 3. A method according to claim 1, wherein theN-terminal extension is 3-15 amino acids in length.
 4. A methodaccording to claim 3, wherein the N-terminal extension is 3-8 aminoacids in length.
 5. A method according to claim 1, wherein thepolypeptide is monoacylated.
 6. A method according to claim 1, whereinone or more amino acid residues in the N-terminal extension are capableof establishing a metal ion complex binding site together with one ormore amino acid residues in the N-terminal end of the polypeptide.
 7. Amethod according to claim 6, wherein said metal ion binding site isderived from: (i) the N-terminal end of porcine or human serum albumin;(ii) the Zn binding site in metalloendopeptidases; or (iii) a Ca⁺²binding enterokinase site from trypsinogen.
 8. A method according toclaim 1, wherein the N-terminal extension comprises at least onenegatively charged amino acid residue that is capable of establishing asalt bridge with the lysine cleavage site N-terminal to the polypeptide.9. A method according to claim 1, wherein the polypeptide is sensitiveto protolytic degradation at its N-terminal end and wherein theN-terminal extension prevents or minimizes such proteolytic degradation.10. A method according to claim 1, wherein the polypeptide has an Ala orPro as the second amino acid residue from the N-terminal end.
 11. Amethod according to claim 10, wherein the desired polypeptide has a Hisas the N-terminal amino acid residue.
 12. A method according to claim 1,wherein the N-terminal extension comprises at least one histidineresidue.
 13. A method according to claim 12, wherein one or morehistidine residues in the N-terminal extension are positioned 1-4residues from the lysine cleavage site.
 14. A method according to claim8, wherein the N-terminal extension comprises at least one Glu or Asp.15. A method according to claim 14, wherein the Glu or Asp residues arepositioned between 1 to 5 residues from the lysine cleavage site.
 16. Amethod according to claim 14, wherein the N-terminal extension comprisesa Glu-Glu-sequence.
 17. A method according to claim 1, wherein theN-terminal extension comprises a sequence selected from the groupconsisting of: Glu-Glu-Ala-His-Lys(SEQ ID NO:1);Glu-(Glu-Ala)₂-His-Lys(SEQ ID NO:2); Glu-(Glu-Ala)₃His-Lys(SEQ ID NO:3);Glu-Glu-Gly-His-Lys(SEQ ID NO:4); Glu-His-Pro-Lys(SEQ ID NO:5);Glu-Glu-Gly-Glu-Pro-Lys(SEQ ID NO:6); Glu-Glu-His-Cys-Lys(SEQ ID NO:7);Glu-Glu-His-His-Lys(SEQ ID NO:8); Glu-His-His-His-Lys(SEQ ID NO:9);Glu-His-Ala-His-Lys(SEQ ID NO:10); Glu-Gly-Ala-His-Lys(SEQ ID NO:11);Glu-His-Gly-His-Gly-Lys(SEQ ID NO:12); Glu-Glu-Ala-His-Glu-Leu-Lys(SEQID NO:13); Glu-Glu-Ala-His-Glu-Ile-Lys(SEQ ID NO:14);Glu-Glu-Ala-His-Glu-Val-Lys(SEQ ID NO:15);Glu-Glu-Ala-His-Glu-Met-Lys(SEQ ID NO:16);Glu-Glu-Ala-His-Glu-Phe-Lys(SEQ ID NO:17);Glu-Glu-Ala-His-Glu-Tyr-Lys(SEQ ID NO:18);Glu-Glu-Ala-His-Glu-Trp-Lys(SEQ ID NO:19);Glu-Glu-Gly-Asn-Thr-Thr-Pro-Lys(SEQ ID NO:20);Glu-Glu-Gly-Asn-Glu-Thr-Glu-Pro-Lys(SEQ ID NO:21),Glu-Glu-Gly-Asn-Asp-Thr-Glu-Pro-Lys(SEQ ID NO:22);Glu-Glu-Gly-Asn-Thr-Thr-Glu-Pro-Lys(SEQ ID NO: 23);Gln-Asp-Ala-His-Lys(SEQ ID NO:24); Glu-Glu-Lys; Asp-Asp-Asp-Asp-Lys(SEQID NO:26); Glu-Glu-Ala-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:29);Glu-Glu-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:30);Leu-Asp-Gly-Arg-Leu-Glu-Ala-Leu-Lys(SEQ ID NO:31);Glu-Glu-Leu-Asp-Gly-Arg-Leu-Glu-Ala-Leu-Lys (SEQ ID NO:32);Glu-Glu-Leu-Asp-Ala-Arg-Leu-Glu-Ala-Leu-Lys(SEQ ID NO:33);Asp-Thr-His-Lys(SEQ ID NO:34); Asp-Ala-His-Lys(SEQ ID NO:35);Glu-His-His-Gly-His-Gly-Lys(SEQ ID NO:36); Asp-Ser-His-Lys(SEQ IDNO:37); Gln-Asp-Thr-His-Lys(SEQ ID NO:38);Glu-Ala-Glu-Ala-Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:39);Glu-Ala-Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:40);Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:41); Trp-His-Trp-Leu-Lys(SEQ IDNO:42); Glu-Glu-Trp-His-Trp-Leu-Lys(SEQ ID NO:43);Glu-Glu-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:44);Glu-Glu-Ala-Glu-Lys(SEQ ID NO:45); Glu-Glu-Gly-Glu-Pro-Lys(SEQ IDNO:46); Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:47);Glu-Ala-Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:48); andGlu-Ala-Glu-Ala-Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:49).
 18. A methodaccording to claim 1, wherein the N-terminal extension has the formula:X_(n) - - - X₁-Lys wherein Lys is a cleavage site and X_(n) - - - X₁ isa peptide sequence of from 2-14 amino acid residues in length having thefunction of preventing or minimizing acylation of the free ε-amino groupin the Lys cleavage and having the further function of protecting theexpressed precursor polypeptide from endoproteolytic cleavage, with theproviso that no X is Lys and that at least one X is His or Glu or Asp.19. A method according to claim 18, wherein: (i) X_(n) - - - X₁ is of2-12 amino acid residues in length; (ii) X_(n) - - - X₁ contains 2-8amino acid residues which are selected from the group consisting of His;Glu; Ala; Asp; Gly; and Pro; (iii) X_(n) - - - X₁ contains 4-10 aminoacid residues which are selected from the group consisting of Glu; Asp;Ala; His; Trp; Tyr; Ile; Val; Met; and Phe; or (iv) X_(n) - - - X₁contains 5-8 amino acid residues selected from the group consisting ofGlu; Asp; Gly; Asn; Thr; Ser; and Pro.
 20. A method according to claim18, wherein the first and second amino acids from the N-terminal end ofthe N-terminal extension are selected from the group consisting of Gluand Asp.
 21. A method according to claim 18, wherein of X_(n) - - -X₁-Lys are selected from the group consisting of Glu-Glu-Ala-His-Lys(SEQID NO:1); Glu-(Glu-Ala)₂-His-Lys(SEQ ID NO:2); Glu-(Glu-Ala)₃His-Lys(SEQID NO:3); Glu-Glu-Gly-His-Lys(SEQ ID NO:4); Glu-His-Pro-Lys(SEQ IDNO:5); Glu-Glu-Gly-Glu-Pro-Lys(SEQ ID NO:6); Glu-Glu-His-Cys-Lys(SEQ IDNO:7); Glu-Glu-His-His-Lys(SEQ ID NO:8); Glu-His-His-His-Lys(SEQ IDNO:9); Glu-His-Ala-His-Lys(SEQ ID NO:10); Glu-Gly-Ala-His-Lys(SEQ IDNO:11); Glu-His-Gly-His-Gly-Lys(SEQ ID NO:12);Glu-Glu-Ala-His-Glu-Leu-Lys(SEQ ID NO:13);Glu-Glu-Ala-His-Glu-Ile-Lys(SEQ ID NO:14);Glu-Glu-Ala-His-Glu-Val-Lys(SEQ ID NO:15);Glu-Glu-Ala-His-Glu-Met-Lys(SEQ ID NO:16);Glu-Glu-Ala-His-Glu-Phe-Lys(SEQ ID NO:17);Glu-Glu-Ala-His-Glu-Tyr-Lys(SEQ ID NO:18);Glu-Glu-Ala-His-Glu-Trp-Lys(SEQ ID NO:19);Glu-Glu-Gly-Asn-Thr-Thr-Pro-Lys(SEQ ID NO:20);Glu-Glu-Gly-Asn-Glu-Thr-Glu-Pro-Lys(SEQ ID NO:21),Glu-Glu-Gly-Asn-Asp-Thr-Glu-Pro-Lys(SEQ ID NO:22);Glu-Glu-Gly-Asn-Thr-Thr-Glu-Pro-Lys(SEQ ID NO: 23);Gln-Asp-Ala-His-Lys(SEQ ID NO:24); Glu-Glu-Lys; Asp-Asp-Asp-Asp-Lys(SEQID NO:26); Glu-Glu-Ala-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:29);Glu-Glu-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:30);Leu-Asp-Gly-Arg-Leu-Glu-Ala-Leu-Lys(SEQ ID NO:31);Glu-Glu-Leu-Asp-Gly-Arg-Leu-Glu-Ala-Leu-Lys (SEQ ID NO:32);Glu-Glu-Leu-Asp-Ala-Arg-Leu-Glu-Ala-Leu-Lys(SEQ ID NO:33);Asp-Thr-His-Lys(SEQ ID NO:34); Asp-Ala-His-Lys(SEQ ID NO:35);Glu-His-His-Gly-His-Gly-Lys(SEQ ID NO:36); Asp-Ser-His-Lys(SEQ IDNO:37); Gln-Asp-Thr-His-Lys(SEQ ID NO:38);Glu-Ala-Glu-Ala-Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:39);Glu-Ala-Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:40);Glu-Ala-Gln-Asp-Thr-His-Lys(SEQ ID NO:41); Trp-His-Trp-Leu-Lys(SEQ IDNO:42); Glu-Glu-Trp-His-Trp-Leu-Lys(SEQ ID NO:43);Glu-Glu-Glu-Ala-Trp-His-Trp-Leu-Lys(SEQ ID NO:44);Glu-Glu-Ala-Glu-Lys(SEQ ID NO:45); Glu-Glu-Gly-Glu-Pro-Lys(SEQ IDNO:46); Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:47);Glu-Ala-Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:48); andGlu-Ala-Glu-Ala-Glu-Ala-Gln-Asp-Ala-His-Lys(SEQ ID NO:49).
 22. A methodaccording to claim 7, wherein the N-terminal extension comprises thesequence His-Z₁—Z₂-Lys(SEQ ID NO:51) wherein Z₁ is Glu; Asp; Asn; Gln;Ser; Thr; Gly; Leu; Ile; Val; Met; Phe or Tyr and Z₂ is Leu; Ile; Val;Met; Phe; Tyr; Trp or Cys.
 23. A method according to claim 7, whereinthe N-terminal extension comprises the sequence Asp-X-His-Lys(SEQ IDNO:50) where X is Ala, Thr or Ser.
 24. A method according to claim 1,wherein the polypeptide belongs to the GRF (growth hormone releasingfactor) family of peptides having a His or Tyr in the N-terminalposition and Ser, Ala or Gly in the next position.
 25. A methodaccording to claim 1, wherein the polypeptide is GLP-1 or GLP-2 or aGLP-1 or GLP2 analogue or derivative.
 26. A method according claim 25,wherein the polypeptide is GLP-1₍₇₋₃₇₎ acylated in position Lys²⁶ andLys³⁴.
 27. A method according to claim 25, wherein the GLP-1 analogue isArg³⁴GLP1₍₇₋₃₇₎acylated in position Lys²⁶.
 28. A method according toclaim 1, wherein the enzymatic cleavage in step (iv) is achieved by useof a lysine-specific endopeptidase.
 29. A method according to claim 1,wherein the host cell is a yeast cell.
 30. A method according to claim29, wherein the yeast cell is a Saccharomyces cerevisiae cell.
 31. Amethod according to claim 30, wherein the yeast cell is a ΔYPS1 cell.32. A method according to claim 1, wherein the acylation step: (i) isperformed in an organic solvent or in a mixture of water and an organicsolvent, wherein the organic solvent is CH₃CN or NMP(N-methyl-pyrrolidon); (ii) is performed in the presence of a metal ion,wherein the metal ion is selected from the group consisting of: Zn, Cu,Co, Ni, Fe, Mg, Mn or Ca; (iv) is conducted at a pH is between 7 and 12;and (v) is performed at a temperature between −5° C. and 35° C.
 33. Aprecursor for a polypeptide, wherein said precursor has the formulaN-terminal-extension-Lys-Z₃—Z₄-*polypeptide*, wherein Lys is a cleavagesite, the N-terminal extension has 2-14 amino acid residues and iscapable of preventing or minimizing acylation of the Lys cleavage siteand protecting the polypeptide precursor against proteolytic degradationduring production of the precursor, Z₃ is His or Tyr and is theN-terminal amino acid residue in the desired polypeptide, Z₄ is the nextamino acid residue from the N-terminal end in the desired polypeptideand is Ala, Ser or Gly, and *polypeptide* is the remaining sequence ofthe desired polypeptide.
 34. A precursor according to claim 33, whereinthe N-terminal sequence is capable of establishing a metal ion complexbinding site together with one or more amino acid residues in theN-terminal end of the polypeptide.
 35. A precursor according to claim33, wherein the N-terminal extension comprises a metal ion binding sitederived from the N-terminal end of albumin.
 36. A precursor according toclaim 33, wherein the N-terminal extension comprises a metal ion bindingsite derived from the Zn binding site in metalloendopeptidases.
 37. Aprecursor according to claim 33, wherein the N-terminal extensioncomprises at least one amino acid residue which is capable ofestablishing a salt bridge with the lysine cleavage site.
 38. Aprecursor according to claim 37, wherein the N-terminal extension iscapable of forming an α-helix.
 39. A precursor according to claim 33,wherein the N-terminal extension comprises a eukaryotic glycosylationsite.
 40. A precursor according to claim 33, wherein the N-terminalextension comprises at least one histidine residue.
 41. A precursoraccording to claim 33, wherein the N-terminal extension comprises atleast one Glu or Asp.
 42. A precursor according to claim 33, wherein thepolypeptide is GLP-1 or GLP-2 or a GLP-1 or GLP2 analogue or derivative.43. A precursor according claim 42, wherein the polypeptide isGLP-1₍₇₋₃₇₎ acylated at position Lys²⁶ and Lys³⁴.
 44. A polypeptideprecursor according to claim 42, wherein the GLP-1 analogue isArg³⁴GLP1₍₇₋₃₇₎acylated at position Lys²⁶.